Glycosulfopeptide inhibitors of leukocyte rolling and methods of use thereof

ABSTRACT

Compounds, compositions and methods for treating conditions characterized by leukocyte rolling are described. The compounds contain glycosulfopeptide structures comprising sulfated tyrosines and sialyated, fucosylated N-acetyllactosamino glycans. The glycosulfopeptides may be conjugated or complexed to other compounds for enhancing serum half-life or for controlled release, for example. Examples of conditions treated include inflammation, ischemia-reperfusion injury, rheumatoid arthritis, atherosclerosis, leukocyte-mediated lung injury, restenosis, and thrombosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 11/239,576,filed Sep. 29, 2005, now U.S. Pat. No. 7,189,828, issued Mar. 13, 2007,which is a continuation of U.S. Ser. No. 10/278,594, filed Oct. 18,2002, now abandoned, which is a continuation-in-part of U.S. Ser. No.09/334,013, filed Jun. 15, 1999, now U.S. Pat. No. 6,593,459, issuedJul. 15, 2003, which claims the benefit of U.S. Provisional ApplicationSer. No. 60/089,472, filed Jun. 16, 1998. U.S. Ser. No. 10/278,594 alsoclaims the benefit of U.S. Provisional Application 60/345,988 filed Oct.19, 2001. Each of the above applications are hereby incorporated byreference herein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This work was supported in part by NIH grants POIHL 54804, HL54502, andAI44902. The U.S. Government has certain rights to this invention.

BACKGROUND

The present invention is directed to glycosulfopeptides and methods oftheir use in treating inflammation and disorders related to leukocyterolling mediated by P-selectin binding.

Inflammation is the reaction of vascularized tissue to local injury.This injury can have a variety of causes, including infections anddirect physical injury. The inflammatory response can be consideredbeneficial, since without it, infections would go unchecked, woundswould never heal, and tissues and organs could be permanently damagedand death may ensue. However, the inflammatory response is alsopotentially harmful. Inflammation can generate pathology associated withrheumatoid arthritis, myocardial infarction, ischemia reperfusioninjury, hypersensitivity reaction, and some types of fatal renaldisease. The widespread problem of inflammatory diseases has fosteredthe development of many “anti-inflammatory” drugs. The idealanti-inflammatory drug would be one that enhances the good effectsresulting from the inflammatory response, and at the same time preventsor reduces the potentially harmful side-effects of this response.

The inflammatory response in regard to blood cells is accompanied byadhesion of circulating neutrophils, the most abundant phagocytic cellin the blood, to activated endothelial cells that line the vessels andmake up the vessel walls. The adherent neutrophils are subsequentlyactivated and the activated neutrophils emigrate from the blood into thesurrounding tissue in a process termed diapedesis. The cells then beginengulfing microorganisms in a process termed phagocytosis and they alsodegranulate, releasing a variety of degradative enzymes, includingproteolytic and oxidative enzymes into the surrounding extracellularenvironment. The mechanisms by which neutrophils adhere, becomeactivated, and emigrate from the blood are currently major topics ofresearch around the world.

Leukocyte recruitment to inflamed tissues is a highly ordered processthat begins with and is to a large extent reliant on selectin-dependentleukocyte rolling. Inhibiting selectin binding therefore holds greatpromise for the treatment of inflammatory diseases and conditions. Theselectin family of adhesion molecules has three functionally andstructurally related members, namely E-selectin (expressed byendothelial cells) L-selectin (expressed by leukocytes) and P-selectin(expressed by endothelial cells and platelets). P-selectin has beenconvincingly implicated in inflammatory disorders includingischemia-reperfusion injury and atherosclerosis. Leukocyte rolling issupported by rapid formation of selectin-selectin ligand bonds at thefront of a cell, coupled with detachment at the rear. With a constantrequirement for new bond formation, leukocyte rolling is thereforesensitive to treatments that block the molecules involved in thisresponse. In keeping with this model, application of antibodies thatblock the selectins or PSGL-1 (P-selectin glycoprotein ligand-1) shouldcause reversal of existing leukocyte rolling in vivo. Chargedpolysaccharides such as fucoidin and dextran sulfate can also inhibitpreexisting leukocyte rolling, presumably by binding to and blocking theselecting.

The realization that the selectin family of adhesion molecules allrecognize sialylated fucosylated glycans, prototypically represented bythe tetrasaccharide sialyl Lewis^(x) (sLe^(x)), fueled development ofcarbohydrate based selectin inhibitors. Data from in vitro bindingassays and from models of inflammation support the notion thatsLe^(x)-mimetic drugs inhibit all three selectins and, as such, shouldbe efficacious against inflammatory disease. Using an intravitalmicroscopy model, where leukocyte rolling was observed immediatelybefore and after application of inhibitors, it was shown that sLe^(x)and close structural mimetics thereof are, in fact, weak inhibitors ofE-selectin dependent rolling and have no impact whatsoever on P- orL-selectin dependent rolling. This fact is consistent with the notionthat sLe^(x) and related structures represent only one component of themacromolecular assemblies that represent true selectin ligands.

The best characterized selectin ligand is PSGL-1, a dimeric mucinpresent on all leukocytes. Studies with antibodies and withgene-targeted mice lacking PSGL-1 have demonstrated that PSGL-1 is themajor ligand for P-selectin dependent leukocyte rolling in themicrocirculation. In addition, it was demonstrated that recombinantPSGL-1 fused to human IgG (rPSGL-Ig) could support rolling interactionsof microspheres with E- and P-selectins in venules and couldcompetitively inhibit leukocyte rolling on E- and L- as well asP-selectin in vivo. However, difficulties of large scale synthesis andfears of immune reactions limit the use of antibodies for therapy,whereas a high possibility of nonspecific side effects limit the use offucoidin and similar agents. Therefore, smaller molecules of definedstructure that selectively bind to selectins with high affinity andwhich prevent binding of selectins to ligands could comprise attractivedrug candidates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show formulas of glycosulfopeptides contemplated by thepresent invention wherein the R groups represented are those in FIGS.5A-5C.

FIGS. 2A and 2B show formulas of alternative embodiments ofglycosulfopeptides contemplated by the present invention wherein the Rgroups are those represented in FIGS. 5A-5C.

FIGS. 3A and 3B show formulas of additional alternative embodiments ofglycosulfopeptides contemplated by the present invention wherein the Rgroups represented are those in FIGS. 5A-5C.

FIGS. 4A and 4B shows specific amino acid sequences for a number ofexemplary glycosulfopeptides contemplated herein, wherein theglycosulfopeptides may comprise from one to three sulfates and R groupsR₁-R₁₅ as defined in FIGS. 5A-5C. In FIGS. 4A and 4B glycosulfopeptide Ais represented by SEQ ID NO:1, B by SEQ ID NO:2, C by SEQ ID NO:3, D bySEQ ID NO:4, E by SEQ ID NO:5, F by SEQ ID NO:6, G by SEQ ID NO:7, H bySEQ ID NO:8, I by SEQ ID NO:9, J by SEQ ID NO:10, K by SEQ ID NO:11, Lby SEQ ID NO:12, M by SEQ ID NO:13 and N by SEQ ID NO:14.

FIGS. 5A, 5B and 5C show chemical structures of a number of R groupswhich are among those which may comprise the glycan portion of theglycosulfopeptides contemplated by the present invention.

FIG. 6 shows four glycosulfopeptides synthesized for further analysis.

FIG. 7 is a graph showing equilibrium affinity binding of 4-GSP-6 tohuman P-selectin at low salt.

FIG. 8 shows the effects of several GSPs on leukocyte rolling in vivo.

FIG. 9 shows the effects of 2-GSP-6 and 4-GSP-6 on leukocyte rollingover a ten minute period.

FIG. 10 shows the effects of 4-GSP-6 on leukocyte rolling velocity at adose of 1.43 μmol/kg.

FIG. 11 shows the effects of 4-GSP-6 on leukocyte rolling velocity at adose of 4.3 μmol/kg.

FIG. 12 shows the effects of 4-GSP-6 on leukocyte rolling velocity at adose of 12.9 μmol/kg.

FIG. 13 shows the clearance rate of 4-GSP from the bloodstream within 10minutes after injection.

FIG. 14 shows the accumulation of 4-GSP-6 in various organs within 10minutes after injection.

FIG. 15 shows schematic structures of A-E of GSPs conjugated in severalways to PEG.

DETAILED DESCRIPTION OF THE INVENTION

The present invention contemplates the use of a new class of syntheticglycosulfopeptides (GSPs) which comprise one or more sulfated tyrosineresidues and a glycan comprising a sialyl Lewis^(x) group or a sialylLewis^(a) group. In a preferred embodiment, the GSPs further comprise anO-glycan comprising a β1,6 linkage to a GaINAc. The present inventioncontemplates methods of using these GSPs in vivo as powerfulanti-inflammatory, antithrombotic, or anti-metastatic compounds whichare able to block the selectin-mediated rolling and adhesion ofleukocytes.

The present invention contemplates use of glycosulfopeptides whichcomprise at least one natural or synthetic amino acid residue able toprovide a glycosidic linkage (e.g., including, but not limited to,serine, threonine, hydroxyproline, tyrosine, hydroxylysine, methionine,lysine, cysteine, asparagine, and glutamine). The peptide backbone ofthe GSP preferably comprises from two amino acids to 30 amino acids, andmore particularly may comprise from 3 to 29 amino acid residues, 4 to 28amino acid residues, 5 to 27 amino acid residues, 6 to 26 amino acidresidues, 7 to 25 amino acid residues, 8 to 24 amino acid residues, 9 to23 amino acid residues, 10 to 22 amino acid residues, 11 to 21 aminoacid residues, 12 to 20 amino acid residues, 13 to 19 amino acidresidues, 14 to 18 amino acid residues, 15 to 17 amino acid residues, or16 amino acid residues.

The glycosulfopeptide contemplated herein preferably comprises at leastone sulfated tyrosine residue, more preferably two sulfated tyrosineresidues, and most preferably three sulfated tyrosine residues. Theglycosulfopeptide contemplated herein may comprise four or five sulfatedtyrosines. Each tyrosine residue is preferably separated by at least oneadditional amino acid residue. The glycosulfopeptide can be constructedby one of the methods described in the specifications of U.S. Ser. Nos.09/849,031, 09/849,562, 09/334,013 filed Jun. 15, 1990 and U.S.Provisional Application 60/089,472 filed Jun. 16, 1998, each of which ishereby expressly incorporated by reference herein in its entirety.

While the invention will now be described in connection with certainpreferred embodiments in the following examples so that aspects thereofmay be more fully understood and appreciated, it is not intended tolimit the invention to these particular embodiments. On the contrary, itis intended to cover all alternatives, modifications and equivalents asmay be included within the scope of the invention as defined by theappended claims. Thus, the following examples, which include preferredembodiments will serve to illustrate the practice of this invention, itbeing understood that the particulars shown are by way of example andfor purposes of illustrative discussion of preferred embodiments of thepresent invention only and are presented in the cause of providing whatis believed to be the most useful and readily understood description offormulation procedures as well as of the principles and conceptualaspects of the invention.

EXAMPLES

As noted, above, the glycosulfopeptides contemplated herein comprise atleast one oligosaccharide conjugated to a linking amino acid on apeptide backbone thereof.

Examples of various oligosaccharides which may comprise the glycan Rgroups of the glycosulfopeptides contemplated for use herein are shownin FIGS. 5A, 5B and 5C. Methods of forming glycosulfopeptides havingthese glycans are shown in U.S. Ser. No. 09/334,013 which has beenexpressly incorporated herein by reference in its entirety. R₁ shown inFIG. 5A is the O-glycan of 2-GSP-6 and 4-GSP-6 shown in FIG. 6.

R₂ is like R₁ except a NeuAc (N-acetylneuraminic acid) group has beenadded in an α2,3 linkage via α2,3-ST (α2,3-sialyltransferase) in thepresence of CMPNeuAc (cystosine monophosphate N-acetylneuraminic acid)to the Gal (galactose) linked to the GaINAc (N-acetylgalactosamine).

R₃ is like R₁ except the Gal has been linked to the GIcNAc(N-acetylglucosamine) in a β01,3 linkage via β1,3-GalT(β1,3-Galactosyltransferase) and Fuc (fucose) has been linked to theGIcNAc in an α1,4 linkage via α1,4-FT (α1,4-Fucosyltransferase).

R₄ is like R₃ except a NeuAc group has been added in an α2,3 linkage viaα2,3-ST in the presence of CMPNeuAc to the Gal linked to the GaINAc.

R₅, R₆, R₇ and R₈ are like R₁, R₂, R₃, and R₄, respectively, except asulfate group has been linked to the GlcNAc.

R₉ and R₁₁are like R₁ and R₇, respectively, except they are lacking aGal in β1,3 linkage to the GalNAc.

R₁₀ is like R₉ but has a sulfate group linked to the GlcNAc.

R₁₂ is like R₁ but has a sialyl Lewis^(x) group in β1,3 linkage to theterminal Gal group.

R₁₃ is like R₁₂ but has a NeuAc in α2,3 linkage to the Gal linked to theGalNAc.

R₁₄ is like R₁₂ except the terminal NeuAc is replaced with a sialylLewis^(x) group in β1,3 linkage to the terminal Gal group.

R₁₅ is like R₁₄ but has a NeuAc in α2,3 linkage to the Gal linked to theGalNAc.

Groups R₁-R₁₅ are merely examples of glycans which may form portions ofthe glycosulfopeptide contemplated herein. It will be understood, by aperson of ordinary skill in the art, that these R groups are onlyrepresentative of the many glycans which may constitute the glycanportion of the glycosulfopeptides of the present invention.

The glycosulfopeptide of present invention in its most basic formcomprises a dipeptide comprising a sulfate group linked to a first aminoacid of the dipeptide and a glycan linked to a second amino acid,wherein the glycan is a sialyl Lewis^(x) group or comprises a sialylLewis^(x) group as a portion thereof. Preferably, the glycan is O-linkedto the peptide. The first amino acid, to which the sulfate is attached,is tyrosine (Tyr). The second amino acid, to which the O-glycan islinked, is preferably a threonine (Thr) or serine (Ser) residue but maybe any other amino acid residue to which an glycan can be linked inO-linkage (for example, tyrosine, hydroxyproline or hydroxylysine).

The present invention further contemplates that the glycan may be linkedin N- or S-linkage to the peptide via an amino acid such as asparticacid, asparagine, glutamic acid, glutamine, arginine, lysine, cysteineor methionine. The present invention contemplates that the peptide maybe covalently derivatized to contain the glycan. Examples of suchdipeptides defined herein are shown as formulas in FIGS. 1A and 1Bwherein “C” represents a threonine, serine, or other residue to whichthe glycan may be linked, and R represents any one of the groups R₁-R₁₅defined herein (and shown in FIGS. 5A-5C, for example). R, of course,may be another glycan not shown in FIGS. 5A-5C if it enables theglycosulfopeptides to function in accordance with the present invention,i.e., to bind with high affinity to P-selectin and inhibit leukocyterolling.

The present invention further contemplates peptides such as thoserepresented as formulas in FIGS. 2A and 2B. Glycosulfopeptides in FIGS.2A and 2B are similar to the glycosulfopeptides represented in FIGS. 1Aand 1B except one or more amino acid residues represented by sequence“B” are positioned between the sulfate-linked residue (tyrosine) and theglycan linked residue “C” (i.e., Ser, Thr or other O-, N-, or S-linkableresidue, natural or derivatized). Sequence B represents any amino acidand k in a preferred embodiment, can number from 0-12 amino acidresidues. Where B=0, the peptides are those shown in FIGS. 1A and 1Babove. Where B=2 or more amino acid residues, the 2 or more residueswhich comprise sequence B may be the same amino acid or different aminoacids.

In one embodiment shown below, the glycosulfopeptide comprises astructure I which comprises a heptapeptide structure having a sulfatedtyrosine residue near the N-terminal end and an O-glycosylated linkingresidue (such as Thr or Ser) near the C-terminal end of the peptide.This GSP comprises five intermediate amino acids represented as X₁, X₂,X₃, X₄, and X₅ as shown below. In one embodiment, X₁ is aspartic acid,X₂ is phenylalanine, X₃ is leucine, X₄ is proline and X₅ is glutamicacid. The heptapeptide may comprise a component (an amino acid orglycosyl component) which distinguishes it from a fragment ofnaturally-occurring or recombinantly expressed forms of PSGL-1, i.e., afragment which could not be obtained from fragmentation of PSGL-1.Alternatively, the GSP may comprise fewer than seven amino acids whereinone or more of X₁-X₅ of structure I is not present. Alternatively, anyone or more of X₁-X₅ may be substituted with a different amino acid,preferably one which has similar functional characteristics as the aminoacid being substituted for. Alternatively, X₁-X₅ may comprise repeats ofthe same amino acid, e.g., five glycine residues. In an especiallypreferred version, the peptide contains one proline residue in theposition between tyrosine and the O-linking residue to which the glycanis linked. In structure I, the O-glycan is R₁ of FIG. 5A.

The glycosulfopeptides represented by formulas in FIGS. 3A and 3B areessentially the same as glycosulfopeptides in FIGS. 2A and 2B excepteach glycosulfopeptide has been extended in an N-terminal and/orC-terminal direction with additional amino acid residues “A” and/or “D”,respectively, where sequence A and sequence D may be, in a preferredversion of the invention, from 0-12 amino acids, and where each sequenceA and sequence D may comprise any amino acid, preferably any naturalamino acid. For example, A and D may each comprise one or more aminoacids which are the same, or may comprise different amino acids,preferably any natural amino acid.

Further, it is contemplated herein that the glycosulfopeptidespreferably comprise more than one sulfated tyrosine residue as shown inFIGS. 4A and 4B. FIGS. 4A and 4B show a number of preferredglycosulfopeptides A-N, each having one, two, or three sulfated tyrosineresidues. Glycosulfopeptides with three sulfated tyrosines areespecially preferred although GSPs having more than three sulfatedtyrosines, for example 4 or 5, are also contemplated herein.Glycosulfopeptides A and H, for example, comprise three tyrosineresidues each having a sulfate group linked thereto. GlycosulfopeptidesB, C, D, I, J, and K each have two sulfated tyrosine residues.Glycosulfopeptides E, F, G, L, M, and N, each have one sulfated tyrosinegroup. The glycosulfopeptides represented in FIGS. 4A and 4B areintended to represent only a subset of the compounds contemplated hereinas will be appreciated by a person of ordinary skill in the art and maybe truncated to have more or fewer amino acid residues or may havesubstituted amino acids as described elsewhere herein, or may have moreamino acids as described elsewhere herein (for example as shown below inTable II).

Preferably, the glycosulfopeptide comprises an O-glycan comprising aβ1,6 linkage to GalNAc. In a particularly preferred embodiment of thepresent invention, the O-glycan of the glycosulfopeptide is core-2based.

In particular, the methods of the present invention contemplate treatingsubjects with glycosulfopeptides having a structure II:

-   -   wherein:        -   Tyr is a tyrosine residue;        -   SO₃ ⁻ is a sulfate group attached to the tyrosine residue;        -   C is an N-, S-, or O-linking amino acid residue;        -   R is a sialylated, fucosylated, N-acetyllactosaminoglycan in            O-, S- or N-linkage to C (for example, one of R₁-R₁₅);        -   A, B, and D represent amino acid sequences each comprising            from 0 to 12 amino acids, with the proviso that the compound            comprises no more than 38 amino acids.

More particularly, A may comprise one or two sulfated tyrosine residues,or B may comprise one or two sulfated tyrosines. The glycosulfopeptidemay have at least one additional sialylated, fucosylated O-, N-, orS-glycan linked to an amino acid residue. The “C” amino acid may be anO-linking amino acid, for example, serine, threonine, hydroxyproline,tyrosine, hydroxylysine, or an N-linking amino acid (e.g., asparagine,lysine, or glutamine) or an S-linking amino acid (such as methionine orcysteine). The R may comprise a β1,6 linkage to a GalNAc. The R groupmay be core-2 based. A Gal of the glycan may have been linked to theGalNAc via a core-1 β1,3-GalT (core-1 β1,3-Galactosyltransferase). Theglycan may have a sialic acid which is neuraminic acid. The glycan mayhave a GlcNAc which is linked to the GalNAc via a β1,6 linkage.

Although N-acetyl neuraminic acid is the preferred sialic acid to beused, other sialic acids which function in a similar manner arecontemplated to be used in the glycosulfopeptides claimed herein. Thesealternative sialic acids include those which can be transferred via theenzyme α2,3-ST, including N-glycolylneuraminic acid, N-acetylneuraminicacid, 9-0-acetyl-N-glycolylneuraminic acid,9-0-acetyl-N-acetylneuraminic acid and other sialic acids described inVarki et al., “Sialic Acids As Ligands In Recognition Phenomena”, FASEBJournal, 11(4): 248-55, 1997, which is hereby incorporated by referenceherein.

The peptide portion of the glycosulfopeptide preferably comprises fromtwo amino acid residues to 30 amino acid residues, and more particularlymay comprise from 3 to 29 amino acid residues, 4 to 28 amino acidresidues, 5 to 27 amino acid residues, 6 to 26 amino acid residues, 7 to25 amino acid residues, 8 to 24 amino acid residues, 9 to 23 amino acidresidues, 10 to 22 amino acid residues, 11 to 21 amino acid residues, 12to 20 amino acid residues, 13 to 19 amino acid residues, 14 to 18 aminoacid residues, 15 to 17 amino acid residues, or 16 amino acid residues.

The invention further contemplates a method of using a glycosulfopeptidecomprising a structure III:X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈-X₁₉-X₂₀-X₂₁-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-X₂₇-X₂₈-X₂₉-X₃₀  (III)wherein:

-   -   X₁ is ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met,        asn, pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₂ is ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met,        asn, pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₃ is ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met,        asn, pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₄ is ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met,        asn, pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₅ is ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met,        asn, pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₆ is ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met,        asn, pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₇ is ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met,        asn, pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₈ is ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met,        asn, pro, gin, arg, ser, thr, val, trp, or tyr; or is absent;    -   X₉ is a sulfated tyr;    -   X₁₀ is ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met,        asn, pro, gin, arg, ser, thr, val, trp, or tyr;    -   X₁₁is a sulfated tyr;    -   X₁₂ is ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met,        asn, pro, gin, arg, ser, thr, val, trp, or tyr;    -   X₁₃ is ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met,        asn, pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₁₄ is a sulfated tyr;    -   X₁₅ is ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met,        asn, pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₁₆ is ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met,        asn, pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₁₇ is ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met,        asn, pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₁₈ is ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met,        asn, pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₁₉ is ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met,        asn, pro, gin, arg, ser, thr, val, trp, or tyr;    -   X₂₀ is a thr, ser, hydroxyproline, tyr, met, hydroxylysine, lys,        cys, asn, or gln having a glycan linked thereto, the glycan        comprising a sialyl Lewis^(x) or sialyl Lewis^(a) group such as,        for example, any one of the R groups defined elsewhere herein;    -   X₂₁ is ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met,        asn, pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₂₂ is ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met,        asn, pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₂₃ ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met, asn,        pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₂₄ ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met, asn,        pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₂₅ ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met, asn,        pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₂₆ ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met, asn,        pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₂₇ ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met, asn,        pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₂₈ ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met, asn,        pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₂₉ ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met, asn,        pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   X₃₀ ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met, asn,        pro, gin, arg, ser, thr, val, trp, or tyr, or is absent;    -   and wherein the glycosulfopeptide has leukocyte rolling        inhibiting activity.

The present invention more particularly comprises a method of using aglycosulfopeptide comprising a structure IV:

-   -   wherein X_(aa1) is an amino acid selected from the group        comprising ala, cys, asp, glu, phe, gly, his, ile, lys, leu,        met, asn, pro, gin, arg, ser, thr, val, trp, or tyr, or is        absent;    -   X_(aa2) is thr, ser, tyr, met, asn, gln, cys, lys,        hydroxyproline, or hydroxylysine, or any N-linking, S-linking or        O-linking amino acid;    -   R is a sialylated, fucosylated, N-acetyllactosaminoglycan in N-,        S- or O-linkage to X_(aa2); and    -   X_(aa3) is an acid selected from the group comprising ala, cys,        asp, glu, phe, gly, his, ile, lys, leu, met, asn, pro, gin, arg,        ser, thr, val, trp, or tyr, or is absent.

Where used herein, a GSP comprising structure IV is intended to mean anyGSP having 38 or fewer amino acids which includes structure IV in wholeor in part.

The present invention more particularly comprises a method of using aglycosulfopeptide comprising a structure V:

-   -   wherein C is thr, ser, tyr, met, asn, gin, cys, lys,        hydroxyproline, or hydroxylysine, or any N-linking, S-linking or        O-linking amino acid; and    -   R is a sialylated, fucosylated, N-acetyllactosaminoglycan in N-,        S- or O-linkage to C.

Where used herein, a GSP comprising structure V is intended to mean anyGSP having 38 or fewer amino acids which includes structure V in wholeor in part, including additional amino acids upstream of the N-terminaltyrosine or downstream of the C-terminal “C” amino acid.

The present invention more particularly comprises a conjugatedglycosulfopeptide and method of its use, the conjugatedglycosulfopeptide comprising a structure VI:

-   -   and a PEG polymer carrier comprising at least one polyalkylene        glycol molecule and a    -   X linking group which conjugates the glycosulfopeptide to the        PEG molecule, the linking group comprising at least one amino        acid selected from the group comprising ala, cys, asp, glu, phe,        gly, his, ile, lys, leu, met, asn, pro, gin, arg, ser, thr, val,        trp, or tyr; and wherein    -   X_(aa1) of VI is thr, ser, tyr, met, cys, asn, gin, lys,        hydroxyproline, or hydroxylysine or any N-linking, S-linking or        O-linking amino acid; and    -   Xaa2 of VI is an amino acid selected from the group comprising        ala, cys, asp, glu, phe, gly, his, ile, lys, leu, met, asn, pro,        gin, arg, ser, thr, val, trp, or tyr, or is absent.

Another X linking group or amino acid could be positioned at anothersite within the peptide backbone of the glycosulfopeptide. The PEGpolymeric carrier may further comprise one or more additional amino acidgroups in linkage to the GSP.

It will be noted that generally the specific amino acids which make upthe peptide backbones of the GSPs described herein (e.g., structuresI-VI) can be substituted with other natural amino acids (except thesulfated tyrosine and R-linking amino acids). Structure VI may comprisefrom 1 to 12 amino acids disposed between the PEG and theN-terminal-most sulfated tyrosine.

More particular, amino acids are substituted with other amino acids fromthe same class. These are referred to as “conservative substitutions”.

By “conservative substitution” is meant the substitution of an aminoacid by another one of the same class; the classes according to Table I.

TABLE I CLASS AMINO ACID Nonpolar: Ala, Val, Leu, Ile, Pro, Met, Phe,Trp Uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln Acidic: Asp, GluBasic: Lys, Arg, His

Non-conservative substitutions (outside each class of Table I) may bemade as long as these do not entirely destroy the effectiveness of theglycosulfopeptide.

The glycosulfopeptides contemplated herein may be produced recombinantlyin an expression system comprising a host cell which has beentransformed to contain a nucleic acid encoding the peptide backbone ofthe glycosulfopeptide and nucleic acids encoding the enzymes necessaryfor expression of the GSP. Transformed host cells such as eukaryoticcells can be cultured to produce the GSPs. The GSPs can be madesynthetically using methods shown in U.S. Ser. No. 09/334,013, which hasbeen expressly incorporated by reference herein.

The invention includes glycosulfopeptide structures presented in TableII (SEQ ID NO.15-48). Each of the amino acids except the sulfatedtyrosine (represented as Styr) and glycosylated threonine (representedas Rthr) may be substituted with any other amino acid, but preferablywith an amino acid from within its own class as shown in Table I. Thethreonine which is glycosylated (Rthr) may be substituted by serine,tyrosine, hydroxyproline, hydroxylysine, methionine, cysteine, lysine,asparagine, or glutamine, for example.

TABLE II gln-ala-thr-glu-Styr-glu-Styr-leu- (SEQ ID NO: 15)asp-Styr-asp-phe-leu-pro-glu-Rthr- glu-pro-pro-glu-met-leuala-thr-glu-Styr-glu-Styr-leu-asp- (SEQ ID NO: 16)Styr-asp-phe-leu-pro-glu-Rthr-glu- pro-pro-glu-met-leugln-ala-thr-glu-Styr-glu-Styr-leu- (SEQ ID NO: 17)asp-Styr-asp-phe-leu-pro-glu-Rthr- glu-pro-pro-glu-metthr-glu-Styr-glu-Styr-leu-asp-Styr- (SEQ ID NO: 18)asp-phe-leu-pro-glu-Rthr-glu-pro- pro-glu-met-leugln-ala-thr-glu-Styr-glu-Styr-leu- (SEQ ID NO: 19)asp-Styr-asp-phe-leu-pro-glu-Rthr- glu-pro-pro-gluglu-Styr-glu-Styr-leu-asp-Styr-asp- (SEQ ID NO: 20)phe-leu-pro-glu-Rthr-glu-pro-pro- glu-met-leugln-ala-thr-glu-Styr-glu-Styr-leu- (SEQ ID NO: 21)asp-Styr-asp-phe-leu-pro-glu-Rthr- glu-pro-proStyr-glu-Styr-leu-asp-Styr-asp-phe- (SEQ ID NO: 22)leu-pro-glu-Rthr-glu-pro-pro-glu- met-leugln-ala-thr-glu-Styr-glu-Styr-leu- (SEQ ID NO: 23)asp-Styr-asp-phe-leu-pro-glu-Rthr- glu-progln-ala-thr-glu-Styr-glu-Styr-leu- (SEQ ID NO: 24)asp-Styr-asp-phe-leu-pro-glu-Rthr- gluala-thr-glu-Styr-glu-Styr-leu-asp- (SEQ ID NO: 25)Styr-asp-phe-leu-pro-glu-Rthr-glu- pro-pro-glu-metthr-glu-Styr-glu-Styr-leu-asp-Styr- (SEQ ID NO: 26)asp-phe-leu-pro-glu-Rthr-glu-pro- pro-glu-metglu-Styr-glu-Styr-leu-asp-Styr-asp- (SEQ ID NO: 27)phe-leu-pro-glu-Rthr-glu-pro-pro- glu-metStyr-glu-Styr-leu-asp-Styr-asp-phe- (SEQ ID NO: 28)leu-pro-glu-Rthr-glu-pro-pro-glu-met ala-thr-glu-Styr-glu-Styr-leu-asp-(SEQ ID NO: 29) Styr-asp-phe-leu-pro-glu-Rthr-glu- pro-pro-gluthr-glu-Styr-glu-Styr-leu-asp-Styr- (SEQ ID NO: 30)asp-phe-leu-pro-glu-Rthr-glu-pro- pro-gluglu-Styr-glu-Styr-leu-asp-Styr-asp- (SEQ ID NO: 31)phe-leu-pro-glu-Rthr-glu-pro-pro-glu Styr-glu-Styr-leu-asp-Styr-asp-phe-(SEQ ID NO: 32) leu-pro-glu-Rthr-glu-pro-pro-gluala-thr-glu-Styr-glu-Styr-leu-asp- (SEQ ID NO: 33)Styr-asp-phe-leu-pro-glu-Rthr-glu- pro-prothr-glu-Styr-glu-Styr-leu-asp-Styr- (SEQ ID NO: 34)asp-phe-leu-pro-glu-Rthr-glu-pro-pro glu-Styr-glu-Styr-leu-asp-Styr-asp-(SEQ ID NO: 35) phe-leu-pro-glu-Rthr-glu-pro-proStyr-glu-Styr-leu-asp-Styr-asp-phe- (SEQ ID NO: 36)leu-pro-glu-Rthr-glu-pro-pro ala-thr-glu-Styr-glu-Styr-leu-asp- (SEQ IDNO: 37) Styr-asp-phe-leu-pro-glu-Rthr-glu- prothr-glu-Styr-glu-Styr-leu-asp-Styr- (SEQ ID NO: 38)asp-phe-leu-pro-glu-Rthr-glu-pro glu-Styr-glu-Styr-leu-asp-Styr-asp-(SEQ ID NO: 39) phe-leu-pro-glu-Rthr-glu-proStyr-glu-Styr-leu-asp-Styr-asp-phe- (SEQ ID NO: 40)leu-pro-glu-Rthr-glu-pro ala-thr-glu-Styr-glu-Styr-leu-asp- (SEQ ID NO:41) Styr-asp-phe-leu-pro-glu-Rthr-gluthr-glu-Styr-glu-Styr-leu-asp-Styr- (SEQ ID NO: 42)asp-phe-leu-pro-glu-Rthr-glu glu-Styr-glu-Styr-leu-asp-Styr-asp- (SEQ IDNO: 43) phe-leu-pro-glu-Rthr-glu Styr-glu-Styr-leu-asp-Styr-asp-phe-(SEQ ID NO: 44) leu-pro-glu-Rthr-glu- ala-thr-glu-Styr-glu-Styr-leu-asp-(SEQ ID NO: 45) Styr-asp-phe-leu-pro-glu-Rthrthr-glu-Styr-glu-Styr-leu-asp-Styr- (SEQ ID NO: 46)asp-phe-leu-pro-glu-Rthr glu-Styr-glu-Styr-leu-asp-Styr-asp- (SEQ ID NO:47) phe-leu-pro-glu-Rthr Styr-glu-Styr-leu-asp-Styr-asp-phe- (SEQ ID NO:48) leu-pro-glu-RthrTable II. Examples of glycosulfopeptides. “Styr” represents sulfatedtyrosine; “Rthr” represents a threonine having a glycan linked thereto.Experimental

A series of glycosulfopeptides (GSPs) were synthesized (FIG. 6). 2-GSP-1(SEQ ID NO.49) and 4-GSP-1(SEQ ID No. 51) each carried onlyN-acetylgalactosamine (GalNAc) on the threonine residue, and hadcysteine and methionine as C-terminal amino acid residues, respectively.2-GSP-6 (SEQ ID No. 50) and 4-GSP-6 (SEQ ID No. 52) were similar to2-GSP-1 and 4-GSP-1, respectively, except each had an R₁ group inO-linkage to the threonine rather than a GalNAc. Positioning of a core-2based O-glycan containing sLe^(x) at a position near to locations ofpotential tyrosine sulfation is critical for high affinity binding.Although absence of sulfate at one or more of three tyrosines on themolecule had a lesser negative impact on binding than absence ofsLe^(x), optimal binding was seen only when each of all availabletyrosines (e.g., three) were sulfated. Equilibrium binding affinity of4-GSP-6 to human P-selectin in vitro at 50 mM NaCl was 71±16 nM (FIG.7).

Experiments were conducted to determine whether derivatives of GSP-6could compete with cell bound selectin ligands and modify leukocyterolling in a physiological setting. Presented here are data whichindicate that glycosulfopeptides 2-GSP-6 and 4-GSP-6 competitivelyinhibit leukocyte rolling in vivo and thus that these and otherglycosulfopeptides have other therapeutic effects in vivo as describedfurther herein below.

Methods and Results

Equilibrium Gel Filtration Chromatograpy

Hummel-Dreyer equilibrium gel filtration experiments were conducted asdescribed. SEPHADEX G-100 columns were equilibrated with buffer and³⁵SO₃-4-GSP-6 (10,000 cpm/ml, specific activity 1700 cpm/pmol).Different amounts of soluble P-selectin were pre-incubated with bufferplus ³⁵SO₃-4-GSP-6 and then added to the column. Samples were elutedwith buffer plus ³⁵SO₃-4-GSP-6 and 140 μI fractions were collected at aflow rate of 70 μ/min. Radioactivity was determined by liquidscintillation counting. Bound GSP and total soluble P-selectin werecalculated from equilibrium gel filtration data by dividing the molaramounts of 4-GSP-6 and soluble P-selectin by the peak volume ofGSP-soluble P-selectin complex.

Animals

C57BL/6 mice were purchased from Harlan (Oxon, UK). Male mice weighingbetween 25 and 35 g were used in these experiments. All procedures wereapproved by the University of Sheffield ethics committee and by the HomeOffice Animals (Scientific Procedures) Act 1985 of the UK.

Intravital Microscopy

The cremaster was prepared for intravital microscopy as described.Briefly, mice were anaesthetized with a mixture of ketamine, xylazineand atropine, cannulations of the trachea, jugular vein and carotidartery were performed, and the cremaster muscle exposed and spread overa specialized viewing platform. Temperature was controlled using athermistor regulated heating pad (PDTRONICS, Sheffield, UK) and thecremaster was superfused with thermocontrolled (36° C.) bicarbonatebuffered saline.

Microscopic observations were made using an upright microscope (NIKONECLIPSE E600-FN, Nikon UK Ltd, UK) equipped with a water immersionobjective (40×/0.80 W). Images were recorded using a CCD camera (DAGEMTI DC-330, DAGE MTI Inc, Michigan City, Ind.) onto sVHSvideo-cassettes. Venules (20-40 μM diameter) were selected and typicallyobserved for the entire experimental period. Centre-line blood flowvelocity (V_(CL)) in was measured in vessels of interest using acommercially available velocimeter (CIRCUSOFT, Hockessin, Del.). Vesselswith V_(CL) between 1 and 5 mm/s were selected for these studies.

Control leukocyte rolling was recorded exactly 30 min after exposure ofthe cremaster muscle for intravital microscopy since leukocyte rollingat this time is almost exclusively P-selectin-dependent. GSPs wereinjected at 31 min and their effects monitored for 10 min. As a positivecontrol, the anti-P-selectin antibody RB40.34 (PHARMINGEN, Oxford, UK)was routinely injected at the end of experiments confirming that rollingwas P-selectin dependent. Blood flow velocities and circulatingleukocyte concentrations were measured at key times (before and aftertreatments) during experiments.

Distribution and Clearance of 4-GSP-6

4-GSP-6 was radioiodinated (¹²⁵I) using iodobeads according tomanufacturer's (PIERCE, Rockford, Ill.) instructions giving specificactivity of 5 mCi/μmol). A mixture of ¹²⁵I-4-GSP-6 (1 μg) and unlabelled4-GSP-6 were injected into mice by the jugular vein at a final dose of4.3 μmol/kg. Blood samples (10 μl) were drawn 1, 2, 4 and 10 min afterinjection of material. Mice were then exsanguinated and urine drainedfrom the bladder into a syringe. Bladder, kidneys, spleen, liver, heart,lungs and brain were also collected. Samples were counted on anautomatic gamma counter (WALLAC 1470, EG&G, Berthold, Milton Keynes, UK)and cpm used to calculate % of injected material located in each of thestudied fluids and organs. Total recovered urine and whole organs werecounted along with samples of blood. The proportion of 4-GSP-6 remainingin the blood was calculated from sample counts assuming a total bloodvolume equivalent to 8% of body weight.

2-GSP-6 and 4-GSP-6 Competitively Inhibit P-Selectin-Dependent LeukocyteRolling In Vivo.

2-GSP-6 and 4-GSP-6 were predicted to compete with cell bound P-selectinligands and inhibit P-selectin-dependent leukocyte rolling in vivo.Intravital microscopy of the mouse cremaster muscle was used toinvestigate this potential. Surgical preparation of mice for intravitalmicroscopy stimulated P-selectin dependent rolling as described.Baseline rolling was observed 30 min after surgery, and GSPs wereinjected at 31 min. Effects of GSPs on the number and velocity ofrolling cells were determined from recordings taken between 32 and 42min after surgery. Both 2-GSP-6 and 4-GSP-6 reduced pre-existingP-selectin dependent leukocyte rolling, whereas 2-GSP-1 and 4-GSP-1 didnot (FIG. 8). These effects could not be attributed to changes in bloodflow or circulating leukocyte counts since blood flow velocity remainedstable throughout observation and systemic leukocyte counts increasedslightly following treatment with either 2-GSP-6 or 4-GSP-6 (Table III).

TABLE III Systematic leukocytes Centerline blood flow (cells/μl blood)velocity (mm/sec) TREATMENT 30 min 32 min 30 min 32 min 2-GSP-6 5840 ±935 6360 ± 1108 3743 ± 493 3777 ± 495 (4.3 μmol/kg) 4-GSP-6 4100 ± 3008300 ± 1100 3865 ± 413 4067 ± 770 (4.3 μmol/kg)Table III. Effects of 2-GSP-6 and 4-GSP-6 on circulating leukocyte countand blood flow in mice with P-selectin-dependent leukocyte rolling.

Interestingly, 2-GSP-6 inhibited P-selectin dependent rolling to agreater extent than 4-GSP-6 although neither compound matched thecomplete inhibition given by the P-selectin blocking antibody (RB40.34).This finding is similar to reported effects of anti-PSGL-1 antibodies,which also reduce P-selectin dependent leukocyte rolling substantiallybut not to the same extent as P-selectin blocking antibodies. Theeffects of 4-GSP-6 were dose-dependent and were maximal at 4.3 μmol/kg(FIG. 8).

The effects of 2-GSP-6 and 4-GSP-6 were significant but short lived.4-GSP-6 caused significant inhibition of leukocyte rolling 1-2 min afterinjection at 4.3 μmol/kg, but this effect was reversed within 2-3 min(FIG. 9). Application of a higher dose (12.9 μmol/kg) of 4-GSP-6 did notincrease the magnitude of inhibition, but did slightly prolong theeffect. In addition to reducing the number of cells rolling through agiven vessel, selectin inhibitors can also increase the velocity ofcells that continue to roll. Although 4-GSP-6 failed to reduce leukocyterolling when given at 1.43 μmol/kg, this dose of the peptide did cause asignificant increase in leukocyte rolling velocity 1 min afterapplication as indicated by a shift to the right of the distribution(FIG. 10). This effect was reversed within 1 min. The increase invelocity caused by 4-GSP-6 at 4.3 μmol/kg was more convincing, but wassimilarly reversed within 1 min (FIG. 11). In contrast, application of4-GSP-6 at 12.9 μmol/kg caused a sustained increase in leukocyte rollingvelocity (FIG. 12). Since surgically-induced rolling develops from apurely P-selectin-dependent response to one that is dependent on both P-and L-selectins between 30 and 60 min, we did not study the duration ofaction of 4-GSP-6 beyond 10 min.

Rapid Clearance of 4-GSP-6

In order to inhibit rolling, selectin antagonists must remain intact atsufficient concentrations in the blood. ¹²⁵I-radiolabelled 4-GSP-6 wasused to investigate the kinetics of clearance from the circulation. Amixture of radiolabelled and unlabelled 4-GSP-6 were injected into thejugular vein giving a final 4-GSP-6 dose of 4.3 μmol/kg. Blood samples(10 μl) were drawn from the carotid artery at serial time points afterapplication of material and counted in a gamma counter. More than 60% ofinjected 4-GSP-6 was cleared from the blood within 1 min of injection(FIG. 13). Following an initial rapid fall in blood concentration, amore gradual clearance is seen between 2 and 10 min. After collection ofthe final blood sample, mice were rapidly killed and various organs andfluids harvested. Approximately 30% of injected 4-GSP-6 can be detectedin the urine within 10 min of its application at 4.3 μmol/kg. SubsequentHPLC analysis demonstrated that 4-GSP-6 was intact in the urine (notshown). Examination of various organs showed little evidence ofpreferential accumulation at sites other than the urine (FIG. 14).

In summary, glycosulfopeptides of the present invention can reversepre-existing, surgically induced leukocyte rolling. This observation isconsistent with a model wherein soluble selectin binding moleculescompete with cell bound ligands preventing the formation of new bondsrequired for continued maintenance of leukocyte rolling. Sincesurgically induced rolling is P-selectin-dependent, these datademonstrate that 2-GSP-6 and 4-GSP-6 are active P-selectin antagonistsin vivo.

Studies with ¹²⁵I-labeled 4-GSP-6 demonstrated that glycosulfopeptidesare rapidly cleared from the circulation (FIG. 13). Only 20% of injectedmaterial remained in the blood 2 min after intravenous injection whereasour first count of rolling flux was made between one and two min. Whenthese factors are accounted for and blood volume of a mouse is estimatedat 8% of body weight, then blood concentration of 4-GSP-6 2 min afterinjection of 4.3 μmol/kg would be approximately 10 μM. Considering thata significant portion of this material may be bound to blood elements,this figure compares quite closely with the dose of 4-GSP-6 required toinhibit neutrophil binding to P-selectin in vitro (4.7 μM).

Design of the clearance studies (blood sampling for 10 min followed bysacrifice and organ harvest) was optimized for detailed tracking of GSPremoval from the blood rather than accumulation elsewhere. Nevertheless,it appears clear that nonconjugated 4-GSP-6 rapidly disappears from theblood and a significant portion of material is cleared to the urinewithin 10 min. Conjugation of the GSP will reduce rate of clearance ofthe GSP (see below). No preferential accumulation in other organsharvested was seen but it was assumed that remaining material isdistributed fairly evenly throughout the body. The fact that a high dose(12.9 μmol/kg) of 4-GSP-6 caused a sustained increase of leukocyterolling velocity suggests that material cleared from the blood and intotissues slowly returns to the blood and is cleared to the urine moregradually. Application via routes other than intravenous as well asother methods described herein may be one way to prolong the activityand reduce clearance of glycosulfopeptides.

It has been recently demonstrated that a recombinant PSGL-1 chimera (arecombinant PSGL-1 fragment fused to IgG, known commercially asrPSGL-Ig) can also competitively reverse existing P-selectin dependentleukocyte rolling. Instantaneous activity of GSPs compares favorablywith that of rPSGL-Ig in that 4.3 μmol/kg (equating to approximately 15mg/kg) gives 50-70% inhibition of leukocyte rolling whereas 30 mg/kgrPSGL-Ig is required for a similar effect. Differences in molecularweight notwithstanding, the activity of the GSP is all the moreremarkable when clearance kinetics are considered (rPSGL-Ig has a halflife of hundreds of hours). Activity of the GSP also compares favorablywith less selective inhibitors such as fucoidin.

Utility

The present invention provides a method for the treatment of a patientafflicted with inflammatory diseases or other such diseases orconditions characterized at least in part by leukocyte rolling whereinsuch disease states or conditions may be treated by the administrationof a therapeutically effective amount of a glycosulfopeptide compound ofthe present invention as described herein to a subject in need thereof.In one embodiment the glycosulfopeptide is SEQ ID NO:53.

The term “inflammation” is meant to include reactions of both thespecific and non-specific defense systems. A specific defense systemreaction is a specific immune system reaction response to an antigen.Examples of a specific defense system reaction include the antibodyresponse to antigens such as rubella virus, and delayed-typehypersensitivity response mediated by T-cells (as seen, for example, inindividuals who test “positive” in the Mantaux test).

A non-specific defense system reaction is an inflammatory responsemediated by leukocytes incapable of immunological memory. Such cellsinclude granulocytes, macrophages, neutrophils, for example. Examples ofa non-specific defense system reaction include the immediate swelling atthe site of a bee sting, the reddening and cellular infiltrate inducedat the site of a burn and the collection of PMN leukocytes at sites ofbacterial infection (e.g., pulmonary infiltrates in bacterialpneumonias, pus formation in abscesses).

Although the invention is particularly suitable for cases of acuteinflammation, it also has utility for chronic inflammation. Types ofinflammation that can be treated with the present invention includediffuse inflammation, traumatic inflammation, immunosuppression, toxicinflammation, specific inflammation, reactive inflammation,parenchymatous inflammation, obliterative inflammation, interstitialinflammation, croupous inflammation, and focal inflammation.

It will be appreciated that the glycosulfopeptides described herein willbe used in methods of diagnosis, monitoring, and treatment ofinflammatory disease processes involving leukocyte rolling includingrheumatoid arthritis, acute and chronic inflammation, post-ischemic(reperfusion) leukocyte-mediated tissue damage, atherosclerosis, acuteleukocyte-mediated lung injury (e.g., Adult Respiratory DistressSyndrome), and other tissue-or organ-specific forms of acuteinflammation (e.g., glomerulonephritis). In other embodiments, theglycosulfopeptides contemplated herein will be used to (1) reducerestenosis in patients undergoing percutaneous coronary interventionssuch as angioplasty and stenting; (2) reduce the sequelae of deep venousthrombosis such as leg swelling, pain, and ulcers; (3) reduce mortalityin patients with myocardial infarction; (4) improve organ transplantsurvival by inhibiting early ischemia-reperfusion injury; (5) reducepulmonary complications and cognitive disorders in patients undergoingheart-lung bypass during coronary artery bypass graft surgery; (6) treatpatients having sickle cell disease.

A therapeutically effective amount of a compound of the presentinvention refers to an amount which is effective in controlling,reducing, or promoting the inflammatory response. The term “controlling”is intended to refer to all processes wherein there may be a slowing,interrupting, arresting, or stopping of the progression of the diseaseand does not necessarily indicate a total elimination of all diseasesymptoms.

The term “therapeutically effective amount” is further meant to definean amount resulting in the improvement of any parameters or clinicalsymptoms characteristic of the inflammatory response. The actual dosewill be different for the various specific molecules, and will vary withthe patient's overall condition, the seriousness of the symptoms, andcounter indications.

As used herein, the term “subject” or “patient” refers to a warm bloodedanimal such as a mammal which is afflicted with a particularinflammatory disease state. It is understood that guinea pigs, dogs,cats, rats, mice, horses, cattle, sheep, and humans are examples ofanimals within the scope of the meaning of the term.

A therapeutically effective amount of the compound used in the treatmentdescribed herein can be readily determined by the attendingdiagnostician, as one skilled in the art, by the use of conventionaltechniques and by observing results obtained under analogouscircumstances. In determining the therapeutically effective dose, anumber of factors are considered by the attending diagnostician,including, but not limited to: the species of mammal; its size, age, andgeneral health; the specific disease or condition involved; the degreeof or involvement or the severity of the disease or condition; theresponse of the individual subject; the particular compoundadministered; the mode of administration; the bioavailabilitycharacteristic of the preparation administered; the dose regimenselected; the use of concomitant medication; and other relevantcircumstances.

A therapeutically effective amount of a compound of the presentinvention also refers to an amount of the compound which is effective incontrolling or reducing an inflammatory response or another conditiondescribed herein dependent at least in part on leukocyte rolling.

A therapeutically effective amount of the compositions of the presentinvention will generally contain sufficient active ingredient (i.e., theglycosulfopeptide portion of the conjugated or non-conjugatedglycosulfopeptide) to deliver from about 0.1 μg/kg to about 100 mg/kg(weight of active ingredient/body weight of patient). Preferably, thecomposition will deliver at least 0.5 μg/kg to 50 mg/kg, and morepreferably at least 1 μg/kg to 10 mg/kg.

Practice of the method of the present invention comprises administeringto a subject a therapeutically effective amount of the activeingredient, in any suitable systemic or local formulation, in an amounteffective to deliver the dosages listed above. An effective,particularly preferred dosage of the glycosulfopeptide (for example,GSP-6, 2-GSP-6 or 4-GSP-6) for substantially inhibiting activatedneutrophils is 1 μg/kg to 1 mg/kg of the active ingredient. The dosagecan be administered on a one-time basis, or (for example) from one tofive times per day or once or twice per week, or continuously via avenous drip, depending on the desired therapeutic effect.

As noted, preferred amounts and modes of administration are able to bedetermined by one skilled in the art. One skilled in the art ofpreparing formulations can readily select the proper form and mode ofadministration depending upon the particular characteristics of thecompound selected, the disease state to be treated, the stage of thedisease, and other relevant circumstances using formulation technologyknown in the art, described, for example, in Remington's PharmaceuticalSciences, latest edition, Mack Publishing Co.

Pharmaceutical compositions can be manufactured utilizing techniquesknown in the art. Typically the therapeutically effective amount of thecompound will be admixed with a pharmaceutically acceptable carrier.

The compounds or compositions of the present invention may beadministered by a variety of routes, for example, orally or parenterally(i.e., subcutaneously, intravenously, intramuscularly,intraperitoneally, or intratracheally).

For oral administration, the compounds can be formulated into solid orliquid preparations such as capsules, pills, tablets, lozenges, melts,powders, suspensions, or emulsions. Solid unit dosage forms can becapsules of the ordinary gelatin type containing, for example,surfactants, lubricants and inert fillers such as lactose, sucrose, andcornstarch or they can be sustained release preparations.

In another embodiment, the compounds of this invention can be tablettedwith conventional tablet bases such as lactose, sucrose, and cornstarchin combination with binders, such as acacia, cornstarch, or gelatin,disintegrating agents such as potato starch or alginic acid, and alubricant such as stearic acid or magnesium stearate. Liquidpreparations are prepared by dissolving the active ingredient in anaqueous or non-aqueous pharmaceutically acceptable solvent which mayalso contain suspending agents, sweetening agents, flavoring agents, andpreservative agents as are known in the art.

For parenteral administration, the compounds may be dissolved in aphysiologically acceptable pharmaceutical carrier and administered aseither a solution or a suspension. Illustrative of suitablepharmaceutical carriers are water, saline, dextrose solutions, fructosesolutions, ethanol, or oils of animal, vegetative, or synthetic origin.The pharmaceutical carrier may also contain preservatives, and buffersas are known in the art.

The compounds of this invention can also be administered topically. Thiscan be accomplished by simply preparing a solution of the compound to beadministered, preferably using a solvent known to promote transdermalabsorption such as ethanol or dimethyl sulfoxide (DMSO) with or withoutother excipients. Preferably topical administration will be accomplishedusing a patch either of the reservoir and porous membrane type or of asolid matrix variety.

As noted above, the compositions can also include an appropriatecarrier. For topical use, any of the conventional excipients may beadded to formulate the active ingredients into a lotion, ointment,powder, cream, spray, or aerosol. For surgical implantation, the activeingredients may be combined with any of the well-known biodegradable andbioerodible carriers, such as polylactic acid and collagen formulations.Such materials may be in the form of solid implants, sutures, sponges,wound dressings, and the like. In any event, for local use of thematerials, the active ingredients usually be present in the carrier orexcipient in a weight ratio of from about 1:1000 to 1:20,000, but arenot limited to ratios within this range. Preparation of compositions forlocal use are detailed in Remington's Pharmaceutical Sciences, latestedition, (Mack Publishing).

Additional pharmaceutical methods may be employed to control theduration of action. Increased half-life and controlled releasepreparations may be achieved through the use of polymers to conjugate,complex with, or absorb the glycosulfopeptide described herein. Thecontrolled delivery and/or increased half-life may be achieved byselecting appropriate macromolecules (for example, polysaccharides,polyesters, polyamino acids, homopolymers polyvinyl pyrrolidone,ethylenevinylacetate, methylcellulose, or carboxymethylcellulose, andacrylamides such as N-(2-hydroxypropyl) methacrylamide, and theappropriate concentration of macromolecules as well as the methods ofincorporation, in order to control release.

Another possible method useful in controlling the duration of action bycontrolled release preparations and half-life is incorporation of theglycosulfopeptide molecule or its functional derivatives into particlesof a polymeric material such as polyesters, polyamides, polyamino acids,hydrogels, poly(lactic acid), ethylene vinylacetate copolymers,copolymer micelles of, for example, PEG and poly(l-aspartamide).

The half-life of the glycosulfopeptides described herein can be extendedby their being conjugated to other molecules such as polymers usingmethods known in the art to form drug-polymer conjugates. For example,the GSPs can be bound to molecules of inert polymers known in the art,such as a molecule of polyethylene glycol (PEG) in a method known as“pegylation”. Pegylation can therefore extend the in vivo lifetime andthus therapeutic effectiveness of the glycosulfopeptide molecule.Pegylation also reduces the potential antigenicity of the GSP molecule.Pegylation can also enhance the solubility of GSPs thereby improvingtheir therapeutic effect. PEGs used may be linear or branched-chain.

PEG molecules can be modified by functional groups, for example as shownin Harris et al., “Pegylation, A Novel Process for ModifyingPhararmacokinetics”, Clin Pharmacokinet, 2001:40(7); 539-551, and theamino terminal end of the GSP, or cysteine residue if present, or otherlinking amino acid therein can be linked thereto, wherein the PEGmolecule can carry one or a plurality of one or more types of GSPmolecules or, the GSP can carry more than one PEG molecule.

By “pegylated GSP” is meant a glycosulfopeptide of the present inventionhaving a polyethylene glycol (PEG) moiety covalently bound to an aminoacid residue or linking group of the peptide backbone of the GSP.

By “polyethylene glycol” or “PEG” is meant a polyalkylene glycolcompound or a derivative thereof, with or without coupling agents orderviatization with coupling or activating moeities (e.g., with thiol,triflate, tresylate, azirdine, oxirane, or preferably with a maleimidemoiety). Compounds such as maleimido monomethoxy PEG are exemplary oractivated PEG compounds of the invention. Other polyalkylene glycolcompounds, such as polypropylene glycol, may be used in the presentinvention. Other appropriate polymer conjugates include, but are notlimited to, non-polypeptide polymers, charged or neutral polymers of thefollowing types: dextran, colominic acids or other carbohydrate basedpolymers, biotin deriviatives and dendrimers, fpr example. The term PEGis also meant to include other polymers of the class polyalkyleneoxides.

The PEG can be linked to any N-terminal amino acid of the GSP, and/orcan be linked to an amino acid residue downstream of the N-terminalamino acid, such as lysine, histidine, tryptophan, aspartic acid,glutamic acid, and cysteine, for example or other such amino acids knownto those of skill in the art. Cysteine-pegylated GSPs, for example, arecreated by attaching polyethylene glycol to a thio group on a cysteineresidue of the GSP.

The chemically modified GSPs contain at least one PEG moiety, preferablyat least two PEG moieties, up to a maximum number of PEG moieties boundto the GSP without abolishing activity, e.g., the PEG moiety(ies) arebound to an amino acid residue preferably at or near the N-terminalportion of the GSP.

The PEG moiety attached to the protein may range in molecular weightfrom about 200 to 20,000 MW. Preferably the PEG moiety will be fromabout 1,000 to 8,000 MW, more preferably from about 3,250 to 5,000 MW,most preferably about 5,000 MW.

The actual number of PEG molecules covalently bound per chemicallymodified GSP of the invention may vary widely depending upon the desiredGSP stability (i.e. serum half-life).

Glycosulfopeptide molecules contemplated herein can be linked to PEGmolecules using techniques shown, for example (but not limited to), inU.S. Pat. Nos. 4,179,337; 5,382,657; 5,972,885; 6,177,087; 6,165,509;5,766,897; and 6,217,869; the specifications and drawings each of whichare hereby expressly incorporated herein by reference.

Alternatively, it is possible to entrap the glycosulfopeptides inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatine-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules), or in macroemulsions. Such techniques are disclosed inthe latest edition of Remington's Pharmaceutical Sciences.

U.S. Pat. No. 4,789,734 describe methods for encapsulating biochemicalsin liposomes and is hereby expressly incorporated by reference herein.Essentially, the material is dissolved in an aqueous solution, theappropriate phospholipids and lipids added, along with surfactants ifrequired, and the material dialyzed or sonicated, as necessary. A reviewof known methods is by G. Gregoriadis, Chapter 14. “Liposomes”, DrugCarriers in Biology and Medicine, pp. 287-341 (Academic Press, 1979).Microspheres formed of polymers or proteins are well known to thoseskilled in the art, and can be tailored for passage through thegastrointestinal tract directly into the blood stream. Alternatively,the agents can be incorporated and the microspheres, or composite ofmicrospheres, implanted for slow release over a period of time, rangingfrom days to months. See, for example, U.S. Pat. Nos. 4,906,474;4,925,673; and 3,625,214 which are incorporated by reference herein.

When the composition is to be used as an injectable material, it can beformulated into a conventional injectable carrier. Suitable carriersinclude biocompatible and pharmaceutically acceptable phosphate bufferedsaline solutions, which are preferably isotonic.

For reconstitution of a lyophilized product in accordance with thisinvention, one may employ a sterile diluent, which may contain materialsgenerally recognized for approximating physiological conditions and/oras required by governmental regulation. In this respect, the sterilediluent may contain a buffering agent to obtain a physiologicallyacceptable pH, such as sodium chloride, saline, phosphate-bufferedsaline, and/or other substances which are physiologically acceptableand/or safe for use. In general, the material for intravenous injectionin humans should conform to regulations established by the Food and DrugAdministration, which are available to those in the field.

The pharmaceutical composition may also be in the form of an aqueoussolution containing many of the same substances as described above forthe reconstitution of a lyophilized product.

The compounds can also be administered as a pharmaceutically acceptableacid- or base-addition salt, formed by reaction with inorganic acidssuch as hydrochloric acid, hydrobromic acid, perchloric acid, nitricacid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organicacids such as formic acid, acetic acid, propionic acid, glycolic acid,lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,maleic acid, and fumaric acid, or by reaction with an inorganic basesuch as sodium hydroxide, ammonium hydroxide, potassium hydroxide, andorganic bases such as mono-, di-, trialkyl and aryl amines andsubstituted ethanolamines.

As mentioned above, the compounds of the invention may be incorporatedinto pharmaceutical preparations which may be used for therapeuticpurposes. However, the term “pharmaceutical preparation” is intended ina broader sense herein to include preparations containing aglycosulfopeptide composition in accordance with this invention, usednot only for therapeutic purposes but also for reagent or diagnosticpurposes as known in the art, or for tissue culture. The pharmaceuticalpreparation intended for therapeutic use should contain a“pharmaceutically acceptable” or “therapeutically effective amount” of aGSP, i.e., that amount necessary for preventative or curative healthmeasures. If the pharmaceutical preparation is to be employed as areagent or diagnostic, then it should contain reagent or diagnosticamounts of a GSP.

All of the assay methods listed herein are well within the ability ofone of ordinary skill in the art given the teachings provided herein.

All references, patents and patent applications cited herein are herebyincorporated herein in their entirety by reference.

The present invention is not to be limited in scope by the specificembodiments described herein, since such embodiments are intended as butsingle illustrations of one aspect of the invention and any functionallyequivalent embodiments are within the scope of this invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings.

1. A glycosulfopeptide compound comprising: a glycosulfopeptide havingup to and including 30 amino acids and comprising the structure (SEQ IDNO:55):

wherein, C is an O-, N-, or S-linking amino acid; and wherein, R is oneof R₁, R₃-R₁₃ or R₁₅ wherein:


2. The glycosulfopeptide compound of claim 1 wherein C is serine,threonine, hydroxyproline, tyrosine, hydroxylysine, methionine,cysteine, lysine, asparagine, or glutamine.
 3. The glycosulfopeptidecompound of claim 1, wherein the glycosulfopeptide is combined with apolyethylene glycol polymer, and wherein the polymer is covalentlylinked to the N-terminal amino acid of the glycosulfopeptide, or to alysine, histidine, tryptophan, aspartic acid, glutamic acid, or cysteineof the glycosulfopeptide, and wherein the polymer has the function ofincreasing the half-life of the glycosulfopeptide compound or controlledrelease of the glycosulfopeptide compound.
 4. A glycosulfopeptidecompound, comprising a glycosulfopeptide combined with a polymer whereinthe polymer is covalently linked to the N-terminal amino acid of theglycosulfopeptide, or to a lysine, histidine, tryptophan, aspartic acid,glutamic acid, or cysteine of the glycosulfopeptide, and wherein thepolymer has the function of increasing the half-life of theglycosulfopeptide compound or controlled release of theglycosulfopeptide compound, the glycosulfopeptide having up to andincluding 30 amino acids and comprising the structure (SEQ ID NO:55):

wherein, C is an O-, N-, or S-linking amino acid; and wherein, R is oneof R₁, R₃-R₁₃ or R₁₅ wherein:


5. The glycosulfopeptide compound of claim 4 wherein C is serine,threonine, hydroxyproline, tyrosine, hydroxylysine, methionine,cysteine, lysine, asparagine, or glutamine.